The present invention relates to a method for establishing a functional adaptive human immune system in a suitable animal model as well as to an animal model produced by the method. The animal model is suitable, for example, for pre-clinical pharmacological studies and the production of human antibodies. Preferably, immunodeficient mice can be used for this purpose that develop hardly any immunological reactions with regard to the transplanted cells.
Basic strategies and technologies of a medical therapy in particular in the field of regenerative medicine are often characterized in animal models (for example, by employing rodents). When applying the principles derived in this way in a clinical application on humans it is often difficult to delimit whether a therapeutic product or a therapeutic strategy is optimal or not for a human target. There are thus two possibilities: a) the human target is invalid in the clinical application or b) the therapeutic agent does not have the same functional properties. This is especially relevant for tumor and transplantation therapies (inter alia, stem cell transplantations) and with particular attention in immune therapies that utilize species-specific monoclonal or polyclonal antibody preparations. Pharmaceutical companies are confronted with the challenge that only 10% of the active ingredients tested in clinical studies will result in a new approved medication. For verifying as well as accepting a new technology or working strategy, pre-clinical models and tools are required in which human cells and tissue ran be examined in a system-biological context that is as complex as possible. Immunodeficiency mice in which human cells of the immune system react with one another without being rejected can provide an answer in this connection because they combine the advantage of a small animal model with an improved correlation to the clinical conditions. Two strategies for developing corresponding animal models in rodents have been realized up to now.
The first strategy utilizes the technology for producing genetically modified (transgenic, knock-out, knock-in) animals by means of introduction of human genes, by induction of specific mutations or the replacement of a murine gene by the homolog human gene in somatic cells or germ cells in order to imitate genetic modifications or in order to express human antigens or targets. The limits of this method, in addition to the high costs and the minimal rate of success, lie in particular in the limited production of usually single human properties and thus their reactivity usually with animal partner molecules as well as usually with non-identical gene expression control mechanisms. Therefore, this method is usually exclusively used for characterization of the potential of active ingredients with regard to active ingredient metabolism and the determination of toxicity or for the examination of activity of an individual target or a set of targets (1; 2).
The second and more complex path is the generation of chimeras by xeno-transplantation (3). This encompasses the administration of human cells or tissue in usually immunodeficient animals. Excellent host animals for generating a human immune system are mouse lines that have several defects in the adaptive immunity such as Rag2−/−/γ−/− (4), BNX or NOD/SCID B2mnull. Different lines of the NOD/SCID (non-obese-diabetic/severe combined immunodeficiency) mouse serve as a standard model for humanization. They are characterized essentially by the following immunodeficiency properties: complete loss of B lymphocytes and T lymphocytes, reduced number of NK cells, defects in the differentiation and function of antigen-presenting cells and the absence of circulating complement. These mice are more susceptible for ionizing radiation than the wild type and have defects in the DNA repair system. The formation of human individual lines or several lines of hematopoiesis in an immunodeficient animal is possible after transplantation of human hematopoietic stem cells, differentiated hematopoietic cells as well as lymphoid organs. As a function of the utilized mouse line and its conditioning as well as the number and the source of hematopoietic stem cells, the development of 0.1 to 90% of human OD45+ cells in peripheral blood or the spleen and bone marrow of animals is possible.
A disadvantage is the extremely high variability of the reconstitution of the hematopoiesis. Moreover, individual human cell types are represented differently, depending on the mouse line and the conditioning of the animals. Characteristic for the NOD/SCID mouse is that preferably and almost exclusively B cells will grow. The human B lymphocytes exhibit the typical pattern of immunoglobulin gene rearrangements (6) and generate a diverse immunoglobulin repertoire after administration of human fetal, umbilical cord blood or adult lymphoid progenitors (7). Circulating T-cells are absent or formed only in an extremely minimal amount and, moreover, are formed with delay. A fast and strong development of the T-lymphocytes is detectable only after pretreatment of the animals with recombinant tumor necrosis factor and the administration of high cell numbers of mononuclear cells (8). The human engraftment however has very high variances within the animal group. The phenotypification of the human T-cells shows, despite the variability, a CD4/CD8 quotient of 1:1 or 1:2 as a function of the employed stem cell source. The cells have a diverse T-cell receptor repertoire. Preliminary functional analyses of these T-cells show in in-vitro proliferation studies with PHA or IL-2 that they can be activated; however, only at a very low level. U.S. Pat. No. 6,627,792 B2 utilizes sections of rib bone tissue of operated patients that is applied subcutaneously into the peritoneal cavity for successful reconstitution of T-cells. Their availability and ethical concerns limit their use. After intravenous injection of individual cell suspensions of this bone tissue, the human chimerism is very low and T-cells could hardly be detected. U.S. Pat. No. 6,060,643 utilizes for reconstitution in BNX mice human hematopoietic cells of G-CSF mobilized peripheral blood (PB), fetal and adult bone marrow (FBM, ABM). Only 17% or 4.7% of the mice were chimeric after 6-8 weeks with an extremely high variability after transplantation of PB or ABM. No chimerism was obtained after administration of FSM. Less than 3% or 15% of the animals receiving transplants of PB or ABM or FBM demonstrated a long-term engraftment. Animals that received PB had an average survival rate of 25% after 30 days. This may be related to the fact that auto-reactive cells were formed or a regulatorily active cell population is not developed. Clear signs of a graft versus host disease were found only after administration of peripheral blood of adult donors (9). This demonstrates the unsatisfactory functional potency of human cells in the context of animal models up to now.
Up to now, human cells of the macrophage dendritic cell type have been detected only as immature CD34− HLA-DR+ CD4+ dendritic cell precursors (DC) after implantation of human mononuclear cells of the umbilical cord blood. No mature DCs could be detected in the lymphoid organs of the NOD/SCID mouse (10). Even though the differentiation of the DCs to the mature cells in the animal model was blocked, the DCs that matured under in-vitro conditions proved to be efficient stimulators in a mixed leucocyte reaction. Cravens et al. (11) demonstrated the development of human mature and reactive DOCs in the NOD/SCID mouse without additional cytokine addition. The cells react to an LPS stimulation in-vitro with a high cytokine secretion of IL-8, TNF-α. IL-10 as well as IL-12p70. Relatively low is the release of IL-1β, IL6 as well as IFNγ. These cytokines however are characteristic in the human system for an acute or systemic infection. Further developments are therefore required in which a cytokine profile that is comparable to the human system must be aimed at.
In summarizing the above, it is apparent that methods and models that exist currently reflect partially the development and maturing of human hematopoietic cells and their reactivity in an animal. However, currently there is no established method that generates a standardized, reproducible model that represents the totality of a human, in particulars adaptive, immune system. However, this is an absolute requirement in order to make testing in preclinical phases more efficient and to minimize therapeutic failure. The preparation as well as the use of human-specific therapeutic or regenerative agents requires a common functional presence of human dendritic cells, T-cells and B-cells, NK-cells, monocytes and granulocytes.